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CoolTip: Low-Temperature Solid-Phase Extraction Microcolumn for Capturing Hydrophilic Peptides and Phosphopeptides.

ABSTRACT: Reversed-phase solid-phase extraction (SPE) techniques are commonly used for desalting samples before LC/MS/MS in shotgun proteomics. However, hydrophilic peptides are often lost during the desalting step under the standard SPE conditions. Here, we describe a simple protocol in which a stop-and-go extraction tip packed with a poly(styrene-divinylbenzene) copolymer disc is used at 4 °C during sample loading without any organic solvent. Using this method, which we designate as the CoolTip protocol, we identified 2.9-fold more tryptic peptides and 6.1-fold more tryptic phosphopeptides from HeLa lysates than the standard SPE protocol for hydrophilic peptides, with a mobile phase of less than 8% acetonitrile in LC/MS/MS. There was no decrease in the recovery of hydrophobic peptides. CoolTip also provided better quantitative reproducibility in LC/MS/MS analysis. We anticipate that this protocol will provide improved performance in many kinds of shotgun proteomics experiments.

SUBMITTER: Ogata K

PROVIDER: S-EPMC8646264 | biostudies-literature |

REPOSITORIES: biostudies-literature

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CoolTip: Low-Temperature Solid-Phase Microextraction Tip for Capturing Hydrophilic Phosphopeptides

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